Method for demonstrating an enzymatic activity of micro-organisms

ABSTRACT

The method consists in the use of one compound of formula: X--NH--R in which X represents one 5-bromoindole-3-yle group and R represents the acyl radical of one amino acid selected between leucine and alanine, as tracer for demonstrating, by the formation of a colored product, a peptidase activity in a culture of micro-organisms.

The present invention relates to a method for demonstrating an enzymaticactivity of microorganisms. Such a method can be used for theidentification of microorganisms which may or may not express thisenzymatic activity.

The detection and identification of micro-organisms are very importantespecially in medicine, in the agrifoods industry, for environmentalcontrol (for example for controlling water, etc.). Micro-organisms maybe desired for their pathogenicity, as contamination indicators, oralternatively for controlling manufacturing processes.

The techniques for detecting and identifying microorganisms arecurrently based on the search for characteristic nucleotide sequences,the search for antigens or antibodies, culturing in a selective ornon-selective medium, or alternatively the search for metabolic andespecially enzymatic activities (for example osidase, esterase,peptidase, oxidase etc. activities).

Usually, the methods for detecting and identifying microorganismscombine several of these techniques. Thus, culturing is used to multiplyand select the desired microorganisms. In order to simplify theirdetection, it has been proposed to demonstrate biochemical activities byintroducing molecules which produce a coloration or a fluorescence,directly into the culture medium. Such media are referred to asdetection media. The biochemical activities can be demonstrated byvarious methods such as:

physicochemical modification of the medium: change of pH revealed usinga coloured or fluorescent indicator (methylumbelliferone),

change of the redox potential revealed using a coloured indicator(tetrazolium salt) or a fluorescent indicator,

reaction of a molecule produced by the microorganisms with a compoundpresent in the medium, leading to a coloration,

hydrolysis of molecules releasing a coloured or fluorescent compound(naphthol, coumarin).

The hydrolyses detected are generally the result of the action of anenzyme produced by the microorganism on a natural or synthetic enzymaticsubstrate. These enzymatic activities are, for example, those of thefollowing enzymes: esterases (for example lipases, phosphatases),osidases (β-galactosidase, β-glucuronidase, N-acetyl-hexosaminidase),peptidases (alanine-aminopeptidase, trypsinase, gelatinase), DNAses,decarboxylases, deaminases, ureases, tryptophanases, oxidases,catalases, etc.

It is known that gelled media are particularly suitable for culturingand isolating microorganisms from a sample, as well as for detecting"target" microorganisms in a mixture of microorganisms. On these media,the microorganisms form colonies that can be detected with the nakedeye, and it is highly desirable for the products of the biochemicalactivities studied to remain localized at their site of production. Thiseffectively makes it possible to distinguish one colony from itsneighbours if they do not express the same activities. Various detectionmethods can thus be used, for example changes in pH (FR-A-2,671,100),esterase activities (FR-2-457,323), osidase activities (FR-A-2,684,110),etc. Needless to say, it is possible to use several of these methods inconjunction, in order to demonstrate several species or strains, and/orin order to increase the sensitivity and/or specificity of thedetection.

There are currently no means available, which are suitable for gelledmedia, for demonstrating the activities of L-alanine-aminopeptidases,D-alanine-aminopeptidases and L-leucine-aminopeptidases ofmicroorganisms. The reason for this is that the enzyme substrates usedto date release coloured or fluorescent molecules which diffuse into thegelled media and/or which are only revealed by UV irradiation (in thecase of naphthylamine or aminocoumarin) and/or after the action ofreagents (in the case of naphthylamine), or whose coloration is ofrelatively poor contrast in the reaction media used in microbiology (inthe case of nitroaniline).

It is known that L-leucine-aminopeptidase has been demonstrated inmammalian histological sections by means of an enzyme substrate,L-leucine-3-(5-bromoindolamine), known as L-Leu-BIA for short, whichproduces a coloured compound after hydrolysis; see Pearson et al., 1963,Lab. Invest., 12: 712, who called this compound L-N-(5-bromoindol-3-yl)(leucinamide hydrobromide). In 1967, Yarborough et al., J.Reticuloendoth. Soc., 4: 390 repeated the technique of Pearson et al. insimilar applications (histological slices). They pointed out that addinga mixture of potassium ferr- or ferrocyanide or copper sulphate inhibitsthe reaction.

In 1975, Lojda and Havrankova, Histochemistry, 43: 355 proposed toimprove the method using the substrate L-Leu-BIA by adding a mixture oftetrazolium salt and phenazine methosulphate, the coloured reactionobserved being derived, in this case, from reduction of the tetrazoliumsalt to formazan.

In the course of the studies which lead to the present invention, aninvestigation was carried out as to whether it was possible to useL-Leu-BIA as an enzyme substrate in the detection of microorganismscultured in particular on gelled media. During preliminary tests,L-Leu-BIA was added to the medium described in Example 1 below. Thismedium is commonly used to search for osidases.

It was not possible to demonstrate a peptidase activity, irrespective ofthe microorganism cultured in this medium (Escherichia coli, Klebsiella,Citrobacter, Pseudomonas, Enterococcus, Staphylococcus, Streptococcus) .

On the other hand, if L-leucine-7-amino-4-methylcoumarin (L-Leu-AMC) isadded to this same medium, a fluorescence is detected with some of thesemicroorganisms. Similarly, in this medium, with osidase substrates(5-bromo-4-chloro-3-indolyl-β-D-galactoside and6-chloro-3-indolyl-β-D-glucuronide), the β-galactosidase andβ-glucuronidase activities of the microorganisms can be detected.Addition of the reagents proposed by Lojda and Havrankova was reflectedby a more or less complete inhibition of the growth of themicroorganisms without allowing the revelation of a peptidase activitywith L-Leu-BIA.

Similarly, with the medium used in Example 2 below, it was not possibleto demonstrate a peptidase activity with L-Leu-BIA, whereas thesubstrate L-Leu-AMC allows this activity to be detected in the samemedium.

It has now been discovered that the absence of results with the BIAderivatives was not due to an incompatibility with the microorganisms orto an inhibition of their multiplication during culturing, but was dueessentially to the conditions of the medium. Indeed, it has beendiscovered that it was possible to reveal the peptidase activity ofmicroorganisms with L-Leu-BIA by using other culture media. The reasonsfor which certain media can be used and others cannot are unknown.Nevertheless, it is possible to determine and develop, by simple routineexperiments similar to those described in the experimental sectionbelow, media and/or ingredients which are suitable or which areunsuitable. The invention thus consisted firstly, in particular, insearching for, and in showing that it was possible to find, culturemedia in which the 5-bromoindolamine-derived peptidase substratesmentioned above can be used to demonstrate the corresponding enzymaticactivities in a microorganism culture.

It has thus been discovered in particular that the following medium canbe used:

    ______________________________________                                        Yeast extract           0.5-25 g/l                                              Gelatin peptone 0.5-25 g/l                                                    NaCl 0-50 g/l                                                                 and optionally:                                                               pH regulator, q.s. pH = 3 to 9                                                and/or:                                                                       Gelling agent 5-35 g/l                                                      ______________________________________                                    

The pH chosen is a pH which is suitable for the microorganism studied.In the case of a gelled medium, the pH is preferably from 5 to 9. The pHcan be adjusted, for example, using hydrochloric acid or sodiumcarbonate.

If L-Leu-BIA is added to such a medium, and inoculation withmicroorganisms is carried out, after culturing, brown or colourlesscolonies are obtained depending on whether the microorganisms do or donot express an L-leucine-aminopeptidase activity.

Comparable results have been obtained with the substrates L-Ala-BIA andD-Ala-BIA.

The subject of the invention is thus the use of at least one compound offormula:

    X--NH--R

in which X represents a 5-bromoindole-3-yl group and R represents theacyl residue of an amino acid chosen from leucine and alanine,

as a tracer which makes it possible to demonstrate, by formation of acoloured product, a peptidase activity in a microorganism culture.

In particular, the group R represents an acyl residue of L-Leu, L-Ala orD-Ala.

The substrates whose use forms the subject of the present application donot inhibit the multiplication of microorganisms in the appropriateculture media. The tracer can thus be used by adding it to themicroorganism culture medium, before the start of culturing or at thestart of culturing.

One of the important advantages of the tracers used according to theinvention is that in the presence of the peptidase activityinvestigated, they give coloured products which do not diffuse into thegelled medium.

They can thus be used in a gelled medium. They can, of course, also beused in a liquid medium.

According to a specific embodiment, at least one other tracer whichmakes it possible to demonstrate, by formation of a coloured orfluorescent product, an enzymatic activity which is generally differentfrom that demonstrated using the compounds of formula X--NH--R asdefined above can also be added to the culture. This can be, forexample, an esterase, osidase or peptidase activity. Additionalinformation can thus be obtained, in association with an absence ofcoloration (or of fluorescence) or in association with a colorationwhich is modified relative to the coloration obtained with only oneenzyme substrate. The tracer chosen will have different properties fromthose of the BIA derivatives: for example, another tracer capable ofgiving a reaction product having a different colour from the browncolour obtained with the BIA derivatives will be chosen. The othertracer (or second tracer) will thus make it possible to reveal, byvirtue of its intrinsic colour, or by virtue of its fluorescence, thepresence of an enzymatic activity for which it is specific. If thepeptidase activity which can be revealed by the BIA derivatives is alsopresent, a modified coloration, different from the said brown colour andalso different from the said intrinsic colour given by the secondtracer, will be obtained. Examples of the use of several substrates, aswell as the information which can be derived therefrom, are given belowin the experimental section. Needless to say, the results can vary witheach species or strain of microorganism studied.

Each case liable to be of interest must thus undergo prior studiesaccording to routine experiments.

The derivatives used to demonstrate various enzymatic activities, andwhich can be used as other tracers, are, in particular, indoxyl,coumarin, resorufin, naphthol, naphthylamine, nitrophenol, nitroaniline,rhodamine, hydroxyquinoline, fluorescein etc. derivatives

Among these derivatives which can be used in combination with the BIAderivatives, mention may be made in particular of5-bromo-4-chloro-3-indolyl-β-D-glucuronide,6-chloro-3-indolyl-β-D-glucoside, L-alanine-7-amino-4-methylcoumarin,4-methylumbelliferyl-N-acetyl-β-D-galactosaminide,resorufin-β-D-galactoside, β-naphthyl sulphate, AS-BI β-D-galactosidenaphthol, L-alanineβ-naphthylamide, o-nitrophenol-β-D-galactoside,carboxybenzoyl-L-arginine-p-nitroanilide, rhodamine-110-bis(L-leucineamide), hydroxyquinoline-β-D-glucoside and fluorescein diacetate.

The subject of the invention is also a process for demonstrating apeptidase activity in a microorganism culture, in which a tracer whichmakes it possible to demonstrate the said activity, by formation of acoloured product, is added to the culture medium of the saidmicroorganisms, characterized in that the said tracer comprises at leastone compound of formula:

    X--NH--R,

in which X and R are defined as above.

The compound X--NH--R can be added before the start of culturing orduring culturing, or even at the end of culturing. The use of the methodof the invention thus involves culturing the microorganisms studied, itbeing understood that this culturing can be carried out before or afteraddition of the compound X--NH--R, and that this addition can also becarried out during culturing. Needless to say, the actual culturing iscarried out by incubation of a suitable culture medium under conditionswhich allow the microorganisms studied to multiply. The compositions ofthe culture media and the culturing conditions which are suitable areknown or can be determined by routine experiments.

The subject of the invention is also a culture medium for microorganismscontaining, besides the necessary ingredients for culturing the saidmicroorganisms, at least one compound of formula X--NH--R.

The derivatives of formula X--NH--R are used at concentrations that aresufficient to give observable coloured reactions. These concentrations,which can be determined by routine experiments, can generally vary from25 to 2000 mg per litre of culture medium.

THE CHARACTERISTICS AND ADVANTAGES OF THE INVENTION ARE ILLUSTRATED BYTHE FOLLOWING EXAMPLES. EXAMPLES Example 1

The culture medium contains, besides water:

    ______________________________________                                        Meat peptone*          15 g/l                                                   Casein peptone** 3 g/l                                                        NaCl 5 g/l                                                                    Tris buffer 0.5 g/l                                                           Na.sub.2 HPO.sub.4 1 g/l                                                      Sodium citrate 1 g/l                                                          Glucose 1 g/l                                                                 Sodium pyruvate 2 g/l                                                         Agar 13 g/l                                                                 ______________________________________                                         *Sold by D.I.F.C.O.                                                           **Sold by D.I.F.C.O.                                                     

L-Ala-BIA, L-Leu-BIA, L-Ala-AMC or L-Leu-AMC is added to this medium atconcentrations of 200 mg/l. The various media obtained, distributed inPetri dishes, are inoculated with microorganisms. The dishes wereincubated at 37° C. for 48 hours. The colonies formed were examinedvisually in ambient light and under a UV lamp (wavelength=365 nm) afterincubation for 24 and 48 hours. The colour or the presence offluorescence were noted. The microorganisms studied were: Escherichiacoli, Klebsiella pneumoniae, Citrobacter freundii, Pseudomonasaeruginosa, Streptococcus agalactiae, Enterococcus faecium,Streptococcus pyogenes, Staphylococcus epidermidis and Candida albicans.The results are presented in Table I below:

                                      TABLE I                                     __________________________________________________________________________    Strains   L-Ala--AMC                                                                           L-Ala--BIA                                                                           L-Leu--AMC                                                                           L-Leu--BIA                                     __________________________________________________________________________    E. coli                                                                              24 h                                                                             .sup. Fluo.sup.1                                                                     .sup. --.sup.2                                                                       Fluo   --                                                48 h Fluo -- Fluo --                                                         K. pneumoniae 24 h Fluo -- Fluo --                                             48 h Fluo -- Fluo --                                                         C. freundii 24 h Fluo -- Fluo --                                               48 h Fluo -- Fluo --                                                         P. aeruginosa 24 h Fluo -- -- --                                               48 h Fluo -- Fluo --                                                         S. agalactiae 24 h -- -- Fluo --                                               48 h Fluo -- Fluo --                                                         E. faecium 24 h -- -- -- --                                                    48 h Fluo -- Fluo --                                                         S. pyogenes 24 h -- -- Fluo --                                                 48 h Fluo -- Fluo --                                                         S. epidermidis 24 h -- -- -- --                                                48 h -- -- -- --                                                             C. albicans 24 h -- -- .sup. NT.sup.3 NT                                       48 h Fluo -- NT NT                                                         __________________________________________________________________________     .sup.1 Fluo = fluorescence                                                    .sup.2 -- = absence of fluorescence and/or absence of coloration              .sup.3 NT = not tested                                                   

The medium used in this example makes it possible to demonstrate theL-alanine-aminopeptidase and L-leucine-aminopeptidase activities withthe reagents L-Ala-AMC and L-Leu-AMC respectively, but when L-Ala-BIAand L-Leu-BIA are used, no hydrolysis is detected.

Example 2

L-Leu-BIA or L-Leu-AMC is added to a 0.1 M, pH 7.3 phosphate buffer at25° C., to concentrations of 400 mg/l. The media obtained weredistributed into microtitration plate wells and inoculated withmicroorganism suspensions. The plates were incubated for 24 hours at 37°C. The wells were examined visually in ambient light and under a UVlamp, as above. After incubation for 24 and 48 hours, the colour of thepresence of fluorescence were noted. The results are presented in TableII below:

                  TABLE II                                                        ______________________________________                                        Strain           L-Leu--AMC L-Leu--BIA                                        ______________________________________                                        E. coli    24 h      .sup. Fluo.sup.1                                                                         .sup. --.sup.2                                   48 h Fluo --                                                                 K. pneumoniae 24 h Fluo --                                                     48 h Fluo --                                                                 C. freundii 24 h Fluo --                                                       48 h Fluo --                                                                 P. aeruginosa 24 h -- --                                                       48 h Fluo --                                                                 S. agalactiae 24 h Fluo --                                                     48 h Fluo --                                                                 E. faecium 24 h -- --                                                          48 h Fluo --                                                                 S. pyogenes 24 h Fluo --                                                       48 h Fluo --                                                                 S. epidermidis 24 h -- --                                                      48 h -- --                                                                 ______________________________________                                         .sup.1 Fluo = fluorescence                                                    .sup.2 -- = absence of fluorescence and/or absence of coloration         

Thus, with the medium used in this example, it is possible todemonstrate the L-leucine-aminopeptidase activity with L-Leu-AMC, butwhen L-Leu-BIA is used, no hydrolysis is detected.

Example 3

The culture medium contains, besides water:

    ______________________________________                                        Yeast extract*         6 g/l                                                    Gelatin peptone** 5 g/l                                                       NaCl 8 g/l                                                                    Na.sub.2 CO.sub.3 0.1 g/l                                                     Agar 13 g/l                                                                 ______________________________________                                         *Sold by Bio Merieux                                                          **Biogelytone sold by Bio Merieux                                        

L-Ala-BIA, L-Leu-BIA, L-Ala-AMC or L-Leu-AMC is added to this medium, toconcentrations of 200 mg/l. The various media obtained, distributed inPetri dishes, are inoculated with the same microorganisms as those usedin Example 1. The dishes are incubated at 370° C. for 48 hours. Thecolonies formed were examined visually in ambient light and under a UVlamp (wavelength=365 nm) after incubation for 24 and 48 hours. Thecolour or the presence of fluorescence were noted. The results arepresented in Table III below:

                                      TABLE III                                   __________________________________________________________________________    Strains   L-Ala--AMC                                                                           L-Ala--BIA                                                                           L-Leu--AMC                                                                           L-Leu--BIA                                     __________________________________________________________________________    E. coli                                                                              24 h                                                                             .sup. Fluo.sup.1                                                                     Brown  Fluo   .sup. --.sup.2                                   Gram-negative 48 h Fluo Brown Fluo Brown                                      K. pneumoniae 24 h Fluo Brown Fluo --                                         Gram-negative 48 h Fluo Brown Fluo Brown                                      C. freundii 24 h Fluo Brown Fluo Brown                                        Gram-negative 48 h Fluo Brown Fluo Brown                                      P. aeruginosa 24 h Fluo Brown -- --                                           Gram-negative 48 h Fluo Brown Fluo Brown                                      S. agalactiae 24 h -- -- Fluo --                                              Gram-positive 48 h Fluo Brown Fluo --                                         E. faecium 24 h -- -- -- --                                                   Gram-positive 48 h Fluo Brown Fluo --                                         S. pyogenes 24 h -- -- Fluo --                                                Gram-positive 48 h Fluo Brown Fluo --                                         S. epidermidis 24 h -- -- -- --                                               Gram-positive 48 h -- -- -- --                                                C. albicans 24 h -- -- .sup. NT.sup.3 NT                                       48 h Fluo Brown NT NT                                                      __________________________________________________________________________     .sup.1 Fluo = fluorescence                                                    .sup.2 -- = absence of fluorescence and/or absence of coloration              .sup.3 NT = not tested                                                   

The medium used in this example makes it possible to demonstrate theL-alanine-aminopeptidase and L-leucine-aminopeptidase activities withL-Ala-AMC and L-Leu-AMC respectively, as well as with L-Ala-BIA andL-Leu-BIA. However, these last two substrates give reactions that areoccasionally delayed. On the other hand, they allow Gram-negativebacteria to be distinguished from Gram-positive bacteria. Indeed:

after incubation for 24 hours with L-Ala-BIA, the Gram-positive bacteriagive no coloration, whereas the Gram-negative bacteria give a browncoloration; and

after 48 hours with L-Leu-BIA, the Gram-positive bacteria give nocoloration, whereas the Gram-negative bacteria give a brown coloration.

Example 4

L-Leu-BIA or L-Ala-BIA was added, alone or incombinationwith5-bromo-4-chloro-3-indolyl-β-D-glucoside (X-Glu),6-chloro-3-indolyl-β-D-glucoside (Z-Glu),4-methylumbelliferyl-β-D-glucoside (MUGI) or5-bromo-4-chloro-3-indolyl-acetate (X-Ac), to the medium of Example 3.

The concentrations of these various substrates are as follows:

L-Leu-BIA: 200 mg/l

L-Ala-BIA: 200 mg/l

X-Glu: 200 mg/l

Z-Glu: 200 mg/l

MuGI: 200 mg/l

These various media, distributed in Petri dishes, are inoculated withmicroorganisms. The strains studied are the same as those in the aboveexamples. The dishes were incubated at 370° C. for 48 hours, and thecolonies formed were examined visually in ambient light and under a UVlamp (wavelength=365 nm). The results of observation of the colour ofthe colonies obtained after incubation for 24 and 48 hours are presentedin Table IV:

                                      TABLE IV                                    __________________________________________________________________________                 L-Ala--BIA           L-Leu--BIA                                               --  X-Glu                                                                             Z-Glu                                                                             MUGI X-Ac                                                                              --  X-Glu                                                                             Z-Glu                                                                             MUGI X-Ac                         Strains Medium No. 1 2 3 4 5 6 7 8 9 10                                     __________________________________________________________________________    E. coli                                                                              24 h  Brown                                                                             Brown                                                                             Brown                                                                             Brown                                                                              Brown                                                                             --  --  --  --   --                           Gram-negative 48 h Brown Brown Brown Brown Brown Brown Brown Brown                                                             Brown Brown-                            grey                                                               K. pneumoniae 24 h Brown Grey Pink- Brown + Grey -- Blue Pink- Fluo                                                            Brown-                       Gram-negative    grey Fluo    grey  grey                                       48 h Brown Grey Pink- Brown + Grey Brown Blue- Pink- Brown + Brown-                                                                grey Fluo brown                                                          grey Grey Fluo grey                                                            C. freundii 24 h                                                             Brown Grey Pink- Brown                                                        + Grey Brown Grey                                                             Pink- Brown + Brown-                                                           Gram-negative    grey                                                        Fluo    grey Fluo grey        48 h Brown Grey Pink- Brown + Brown Brown Grey Pink- Brown + Brown-                                                                grey Fluo    grey                                                        Fluo grey                    P. aeruginosa 24 h Brown Brown Brown Brown Brown- -- -- -- --                 Gram-negative      grey                                                        48 h Brown Brown Brown Brown Brown- Brown Brown Brown Brown Brown-                                                                   grey     grey                                                           S. agalactiae 24 h --                                                        -- -- ---- -- -- -- --                                                        --                            48 h Brown Brown Brown Brown Grey -- -- -- -- --                             E. faecium 24 h -- Blue Pink Fluo Blue -- Blue Pink Fluo Blue                  48 h Brown Blue- Pink Brown + Blue- -- Blue Pink Fluo Blue-                     grey grey Fluo grey     grey                                               S. pyogenes 24 h -- -- -- -- -- -- -- -- -- --                                 48 h Brown Brown Brown Brown Blue- -- -- -- -- --                                  grey                                                                    S. epidermidis 24 h -- -- -- -- -- -- -- -- -- --                              48 h -- -- -- -- Tur- -- -- -- -- --                                               quoise                                                                __________________________________________________________________________

On media 2, 3 and 4, it is possible, after incubation for 24 hours, todistinguish the Enterococcus bacteria (E. faecium) which are the onlyones to give a coloration other than brown or grey. Media 2, 3 and 4also make it possible, after culturing for 24 hours, to distinguish theGram-positive bacteria, which show either an absence of coloration or acoloration other than brown or grey. Media 7, 8 and 9 allow similardistinctions, but after incubation for 48 hours. On medium 5 afterincubation for 48 hours, only S. epidermidis gives turquoise-colouredcolonies, the other bacteria giving brown to blue-grey colonies.

It should be pointed out that when the substrates below are presentalone in the reaction medium, the presence of an osidase and theresulting hydrolysis of the 5-bromo-4-chloro-3-indolyl-β-D-glucoside(X-Glu) leads to the appearance of a turquoise-blue colour, thehydrolysis of 6-chloro-3-indolyl-β-D-glucoside (Z-Glu) leads to theappearance of a purple-pink colour. The hydrolysis of4-methylumbelliferyl-β-D-glucoside (MUGI) leads to the appearance of ablue fluorescence under a UV lamp (wavelength=365 nm) and, in thepresence of an esterase, the resulting hydrolysis of5-bromo-4-chloro-3-indolyl-acetate (X-Ac) leads to the appearance of aturquoise-blue colour.

Example 5

The culture medium contains, besides water:

    ______________________________________                                        Yeast extract*         6 g/l                                                    Gelatin peptone** 5 g/l                                                       NaCl 8 g/l                                                                    Na.sub.2 CO.sub.3 0.1 g/l                                                   ______________________________________                                         *Sold by Bio Merieux                                                          **Biogelytone sold by Bio Merieux                                        

L-Leu-BIA or L-Ala-BIA is added, alone or combined together, or incombination with 5-bromo-4-chloro-3-indolyl-acetate (X-Ac) to thismedium.

The concentrations of these various substrates are as follows:

L-Leu-BIA: 300 mg/l

L-Ala-BIA: 300 mg/l

X-Ac: 200 mg/l

The media obtained were distributed into microtitration plates andinoculated with microorganism suspensions, as above. The plates wereincubated for 48 hours at 37° C. The colours of the wells obtained afterincubation for 24 and 48 hours are presented in Table V:

                                      TABLE V                                     __________________________________________________________________________                             L-Ala--BIA +                                             L-Leu--BIA L-Ala--BIA L-Leu--BIA L-Ala--BIA + X-Ac                          Strains Medium No. 1 2 3 4                                                  __________________________________________________________________________    E. coli                                                                              24 h  --    Brown Brown  Grey                                             48 h Brown Brown Brown Grey                                                  K. pneumoniae 24 h Brown Brown Brown Grey                                      48 h Brown Brown Brown Grey                                                  C. freundii 24 h Brown Brown Brown Grey                                        48 h Brown Brown Brown Grey                                                  P. aeruginosa 24 h Brown Brown Brown Grey                                      48 h Brown Brown Brown Grey                                                  S. agalactiae 24 h -- -- Brown Blue-grey                                       48 h -- Brown Brown Blue-grey                                                E. faecium 24 h -- -- Brown Blue-grey                                          48 h -- -- Brown Blue-grey                                                   S. pyogenes 24 h -- -- Brown Blue-grey                                         48 h Brown Brown Brown Grey-brown                                            S. epidermidis 24 h -- -- -- Turquoise                                         48 h -- -- -- Turquoise                                                    __________________________________________________________________________

It is seen that with the media 3 and 4, it is possible to distinguish S.epidermidis from the other bacteria. With medium 1, it is possible todistinguish the Gram-negative bacteria from the Gram-positive cells(except for S. pyogenes) after incubation for 48 hours. Medium 2 allowsdifferentiation of the Gram-positive and Gram-negative bacteria afterincubation for 24 hours.

Example 6

The culture medium contains, besides water:

    ______________________________________                                        Beef extract*          3 g/l                                                    Gelatin peptone** 5 g/l                                                       NaCl 8 g/l                                                                    Agar 15 g/l                                                                 ______________________________________                                         *Sold by Bio Merieux                                                          **Biogelytone sold by Bio Merieux                                        

L-Ala-BIA or L-Ala-AMC is added to this medium, to concentrations of 300mg/l and 200 mg/l respectively. The various media obtained, distributedinto Petri dishes, are inoculated with microorganisms. The dishes areincubated at 370° C. for 48 hours. The colonies formed were examinedvisually in ambient light and under a UV lamp as above, after incubationfor 24 and 48 hours. The colour or the presence of fluorescence werenoted. The results are presented in Table VI below.

                  TABLE VI                                                        ______________________________________                                        Strain           L-Leu--AMC L-Leu--BIA                                        ______________________________________                                        E. coli    24 h      .sup. Fluo.sup.1                                                                         Brown                                            48 h Fluo Brown                                                              K. pneumoniae 24 h Fluo Brown                                                  48 h Fluo Brown                                                              C. freundii 24 h Fluo Brown                                                    48 h Fluo Brown                                                              P. aeruginosa 24 h Fluo --                                                     48 h Fluo Brown                                                              S. agalactiae 24 h .sup. --.sup.2 --                                           48 h Fluo --                                                                 E. faecium 24 h -- --                                                          48 h Fluo --                                                                 S. pyogenes 24 h -- --                                                         48 h Fluo Brown                                                              S. epidermidis 24 h -- --                                                      48 h -- --                                                                   C. albicans 24 h -- --                                                         48 h Fluo Brown                                                            ______________________________________                                         .sup.1 Fluo = fluorescence                                                    .sup.2 -- = absence of fluorescence and/or absence of coloration         

With the medium used in this example, it is thus possible to demonstratethe L-alanine-aminopeptidase activity with L-Ala-AMC as well as withL-Ala-BIA.

I claim:
 1. A microorganism culture medium containing, besides theingredients necessary for culturing microorganisms, at least onecompound of formula:

    X--NH--R

in which X represents a 5-bromoindol-3-yl group and R represents theacyl residue of an amino acid chosen from leucine and alanine.
 2. Theculture medium according to claim 1, containing from 25 to 2000 mg/l ofthe said compound.
 3. The culture medium according to claim 1, whereinthe culture medium is a gelled medium.
 4. A method for demonstrating apeptidase activity in a microorganism culture, wherein a tracer, whichmakes it possible to demonstrate said peptidase activity by formation ofa colored product, is added to a culture medium of microorganisms, saidtracer comprising at least one compound of formula:

    X--NH--R

in which X represents a 5-bromoindol-3-yl group and R represents an acylresidue of an amino acid chosen from leucine and alanine.
 5. The methodaccording to claim 4, wherein R represents an acyl residue of L-Leu,L-Ala or D-Ala.
 6. The method according to claim 4, wherein said culturemedium is a gelled medium.
 7. The method according to claim 4, whereinsaid culture medium is a liquid medium.
 8. The method according to claim4, wherein at least one other tracer is added to the culture medium,wherein said at least one other tracer makes it possible to demonstrate,by formation of a colored or fluorescent product, an enzymatic activitydifferent from said peptidase activity.